The present invention relates to a method for quantitatively measuring an immunosystem reactant, specifically, an antigen or an antibody. More particularly, the present method is an ultrasensitive enzymatic radioimmunoassay method.
Various methods are known to quantitatively and qualitatively measure antigens and antibodies in complex mixtures and solutions such as body fluids from humans and other animals, and laboratory and environmental specimens. Antigens can include viruses, bacteria, parasites, hormone toxins, tumor markers, drugs, and the like. The measurements are useful for diagnostic, prognostic, epidemiological, and experimental purposes.
Solid-phase radioimmunoassay methods are known in the art. These assays have been especially useful for the detection of immunosystem reactants. In general, a radioimmunoassay comprises preparing a solid substrate, selectively coupling the immunosystem reactant to the substrate, and then coupling a radiolabelled antibody to the reactant. After washing away any uncoupled radiolabelled antibodies, the radioactivity of the coupled antibody is measured by radioactive counting methods, for example, gamma or scintillation counting. The counting yields a quantitative determination of the amount of immunosystem reactant bound to the solid substrate. Generally, the radiolabelled antibody is labelled with the radioisotope iodine.sup.131 or iodine.sup.125. Generally, radioimmunoassay (RIA) can measure down to 10.sup.-12 grams per milliliter of material being tested for.
RIA is subject to certain disadvantages. Since the actual antibody is radiolabelled, either the solution must be made up shortly before use or a large stock of various different solutions must be held in inventory. The short half-life of the isotopes utilized limits the shelf life of the reagents. It is known that iodine.sup.131 and iodine.sup.125 subject the users of RIA to a definite biohazard. Radioiodine is expensive. The radioactive labelling of any compound can cause a number of secondary changes in the compound that may affect its properties and its behaviour in an assay system. If the radionuclide used for the labelling is an isotope of an element already present in the native unlabelled molecule and if the labelling involves only the simple substitution of the radioactive isotope for the non-radioactive one, then the labelled compound might be assumed to have the identical chemical and immunologic properties as the unlabelled. However, this ideal situation is seldom realized.
The decay of the radioactive label releases a certain amount of energy that is much higher than the energy involved in the chemical bonds that maintain the molecular structure of the compound. Therefore, decay can easily break adjacent bonds and cause disintegration of the molecular structure. Additionally, when a new element is formed from the decay of the radiolabel, the structure of the original molecule can be altered to the extent that the molecule will disintegrate or no longer serve the purpose for which it is intended. If the labelled molecule contains only a single radioactive atom, the problem that the molecule remaining after a decay event will not be the site of any further decay events leaves that molecule unlabelled and hence unmeasurable. Thus, the useful period of a material is shorter than could be expected from the simple half-life of the radioisotope alone.
If it is necessary to perform labelling by introducing a radioactive isotope that is not a normal component of the unlabelled material, not only will radiation change the material, but the chemical composition of the radio-labelled material may be different from that of unlabelled material. It has been shown that iodination as such can influence the potency of biologically active material. Additionally, the chemical properties of the radiolabelled material may be altered not only by the presence of a radioactive element that is not a normal constituent of the native molecule but also by the procedures used to introduce the radiolabel into the compound. Thus, RIA is subject to a number of disadvantages.
Enzyme immunoassays (also known as EIA and as enzyme-linked immunosorbent assays or ELISA) have been developed in an attempt to overcome the problems associated with RIA. ELISA is similar in design to solid-phase RIA described above except that an enzyme is utilized as the antibody marker instead of the radioisotope. This enzyme-antibody conjugate is bound to the solid-phase substrate by a series of antibody-antigen reactions and is utilized to convert an indicator material to produce a visible color. This color is measured spectrophotometrically. The fact that a single molecule of enzyme is capable of reacting with a large number of indicator molecules provides for amplification of the results and thus provides a high degree of sensitivity. An example of a suitable enzyme is alkaline phosphatase which can be used with p-nitrophenylphosphate which when hydrolyzed liberates a yellow p-nitrophenolate which is measured in a spectrophotometer at 400 nanometers. ELISA has the advantage over RIA in that the reagents that are used, namely the enzyme-conjugated antibody has a longer self life, is not subject to radiodecay effects and does not pose much of a biohazard to laboratory workers.
In practice, however, the sensitivity of ELISA has not significantly exceeded that of RIA. Specifically, ELISA can give results down to 10.sup.-13 grams per milliliter of material being tested.